Mechanism of gene transcription and its regulation will be studied in polyoma virus (PV). The regulatory region of the virus is located between the coding sequences of two sets of genes transcribed divergently from presumably overlapping promoters early and late in infection. The primary objectives of the proposed project are to define the elements of individual promoter and to understand the regulation of transcription of these genes. The latter involves specific sets of DNA-protein and protein-protein interactions among promoter DNA sequences, cellular RNA polymerase IL, cellular transcription factors and the viral regulatory protein T antigen. The objectives will be achieved by introducing mutations systematically in the promoter region by in vitro procedures and by testing the promoter activity in vivo and, in vitro in Hela Cell extracts. For in vivo assay, the promoter will be linked to the chloramphenicol acetyltransferase (CATase) gene of E. coli to make the CATase in the mammalian cell where it is normally absent. The amount of the enzyme synthesized will be taken as a measure of the promoter activity. Different cell lines, such as mouse (3T6), undifferentiated embryonal carcinoma (PCC4, F9) and differentiated cells (Friend erythroleukemic cell) will be used for in vivo characterization of the mutants. The "optimal" PV enhancer structure selected as the one best responding to the cellular factor(s) in these cell lines will be defined. The mechanism of regulation of the early transcription and that of the activation of late transcription by T antigen will be studied in vivo and in vitro. Two possible models that will be tested for the switch-over mechanism from "early- early" to the "late-early" or from the "early" to "late" transcription are a) whether this shift is a direct effect of T antigen binding to the regulatory region b) whether T antigen plays an indirect role because of its role in DNA replication. The cellular factor(s) involved in transcription will be identified by competition transcriptional and DNA binding assay and their points of contact on the template, will be determined by DNase protection experiments in vitro and in vivo, in absence or presence of T antigen. Experiments parallel to those conducted in vitro with naked DNA as template will be carried out with PV minichromosome reconstituted in vitro or isolated from infected cells or from mature virions. The possibility of sequence-directed or protein-induced bending of the regulatory region will be tested.